Phase Contrast Microscopy

Abstract

Phase contrast microscopy is a method that enables us to see very transparent objects, which are otherwise almost invisible
by ordinary light microscopy, in clear detail and in good contrast to their surroundings. This is achieved optically, without
altering the specimen by staining or other processing.

All parts of this figure show the same field of view of living HeLa cells (a–e) and fixed, embedded HeLa cells in thin section
(f–h).

(a) Living HeLa cells in culture by phase contrast. (b) The same cells by transmitted‐light bright‐field microscopy. (c) In
bright‐field mode, without phase contrast, closing the condenser diaphragm will enhance contrast to some degree, but at the
expense of resolution in the image. This method is to be avoided. (d) Same image as (a), but the image has been taken with
the annulus and phase plate out of alignment (see also Figures 3e,f). (e) The use of a green filter improves the quality of
the phase contrast image. (f) Stained HeLa cells, together with the bright‐field image (g) for comparison with the phase contrast
image (h).

Parts (h) and (i) are included for comparison of phase contrast images of living cells with those that have been fixed, embedded
and sectioned thinly. The manner in which cells and tissues are fixed (if at all) and prepared will influence the resulting
phase contrast image. The living cells (h) exhibit high contrast, where there is a relatively high difference of refractive
index between the cells and the watery medium they are contained in. The sections of cells embedded in resin in (i) exhibit
lower contrast. This is because there is a smaller difference of refractive index between the cell constituents and the background
resin. Likewise, cells fixed in methanol, an extracting fixative, exhibit a higher contrast image than those fixed in paraformaldehyde,
a crosslinking fixative that retains more of the cytoplasm.

Figures (a)–(e) were taken using a Zeiss Axiovert 25, inverted microscope for tissue culture using a 32 × NA 0.5 long working
distance objective. Figures (f)–(h) were taken using a Zeiss Axiophot microscope equipped with a Plan Neofluar 40 × NA 1.30
oil immersion phase contrast objective.

Figure 2.

Ray diagram of the phase contrast method. The heavy lines represent the undiffracted beams, while the diffracted beams are
shown by dashed lines. Adapted with permission from Plášek and Reischig .

Figure 3.

(a) and (b) show the top view of different types of phase contrast condenser, in which the various annuli are contained within
a housing. This permits them to be changed quickly and efficiently as required. (c) The commonly encountered green inscription
engraved on the barrel of a phase contrast objective. The correct annulus to use is denoted, shown here by the designation
Ph3. (d) The underside of the condenser in (b), revealing the separate controls for centring the condenser onto the optical
axis during alignment of the microscope, and those for independently aligning the annulus with the phase ring. The different
sizes of annuli can also be seen. (e) and (f) show the effects on the phase contrast image of not having the annulus and phase
ring in absolute alignment.